Bacillus thuringiensis has been used as a microbial insecticide for over twenty years. Its insecticidal activity resides in proteinaceous crystalline inclusions which are produced during sporulation. When ingested by susceptible insect larvae, they are dissolved in the intestinal lumen and proteolytically processed to active toxins which cause lysis of midgut epithelial cells.
Brush border membrane vesicles (BBMV) from larval midgut are very useful in analyzing the mode of action of insecticidal bacterial endotoxins. Indeed, a receptor binding assay which measures the binding of the endotoxin to brush border membrane vesicles has been developed.
For example, Hofmann et al. reported the binding of endotoxin of B. thuringiensis subspecies thuringiensis to brush border membrane vesicles of Pieris brassicae larval midgut C. Hofmann, P. Luthy, R. Hutter and V. Pliska, Eur. J. Biochem. 173, pp. 85-91 (1988). See also B. H. Knowles and D. J. Ellar, J. Cell Sci. 83, pp. 89-101 (1986); C. Hofmann, H. Vanderbruggen, H. Hofte, J. Van Rie, S. Jansens and H. Van Mellaert, Proc. Natl. Acad. Sci. USA 85, pp. 7844-48 (1988); J. Van Rie, S. Jansens, H. Hofte, D. Degheele and H. Van Mellaert, Appl. Environ. Microbiol. 56, pp. 1378-85 (1990); M. G. Wolfersberger, Experientia 46, pp. 475-77 (1990); J. Van Rie, W. H. McGaughey, D. E. Johnson, B. D. Barnett and H. Van Mellaert, Science 247, pp. 72-74 (1990); and S. C. MacIntosh, T. B. Stone, R. S. Jokerst and R. L. Fuchs, Proc. Natl. Acad. Sci. USA 88, pp. 8930-33 (1991).
Brush border membrane vesicles have also been utilized for studies of cotransport mechanisms in lepidopteran gut. See, e.g., B. Giordana, V. F. Sacchi, P. Parenti and G. M. Hanozet, Am. J. Physiol. 257, pp. R494-R500 (1989); G. M. Hanozet, B. Giordana and V. F. Sacchi, Biochem. Biophys. Acta 596, pp. 481-86 (1980); M. Wolfersberger, P. Luthy, A. Maurer, P. Parenti, V. F. Sacchi, B. Giordana and G. M. Hanozet, Comp. Biochem. Physiol. 86A, pp. 301-08 (1987); and V. F. Sacchi, P. Parent, G. M. Hanozet, B. Giordana, P. Luthy and M. G. Wolfersberger, FEBS Lett. 3904, pp. 213-18 (1986).
However, research into the mode-of-action of stomach active insecticides and studies of cotransport mechanisms in lepidopteran gut is presently limited. The experiments require large amounts of brush border membrane vesicles which are not widely available partly due to the difficulty in isolating them from insect midguts. The current method for isolating brush border membrane vesicles requires dissecting individual insect larvae to obtain the midgut tissue. This is very time consuming and labor intensive.
Moreover, most biochemical studies of insect midgut function and mode-of-action have focused on lepidopteran systems using relatively large species, e.g., Manduca sexta, Heliocoverpa virescens and Spodoptera fruqiperda. Comparatively little research has been done on either coleoptera or diptera, partially due to the difficulty in isolating brush border membrane vesicles from small insects. Large insects allow for relatively easy dissection with substantial yield of brush border membrane vesicles per insect, but most lepidopterans are much smaller. On the other hand, small lepidopteran larvae are very difficult to dissect, requiring large number of larvae for isolating a relatively small quantity of brush border membrane vesicles.
A diversity of insect model systems will enhance the overall understanding of Bacillus thuringiensis mode-of-action and, therefore, enhance our ability to improve these insecticides.
It is therefore an object of the present invention to provide an improved method for isolating brush border membrane vesicles from insect larvae which produces large quantities of vesicles in a short time.
It is another object of the present invention to provide a method for isolating brush border membrane vesicles from small insect larvae.